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The steroidogenic responsiveness of adrenal cell suspensions prepared from domestic chicken adrenal tissue at the end of embryogenesis was compared to the responses of similar preparations derived from the wild and domesticated mallard duck (Anas platyrhynchos), and the domesticated muscovy duck (Cairina moschata). In all cases, the masses of corticosterone (B), aldosterone (Aldo), and deoxycorticosterone (DOC) released from cells incubated in medium containing 1-24 ACTH exceeded the estimated hormone content of the freshly dispersed cells; the induced rates of corticosteroid release were, therefore, presumed to reflect de novo hormone synthesis. When chicken cells were incubated in medium containing 1-24 ACTH, there were progressive, dose-dependent increases in B and DOC synthesis over a range of concentrations spanning two orders of magnitude; only small, non-dose-related, albeit significant, increases in Aldo release were observed. The 1-24 ACTH-induced increases in B and Aldo synthesis by the mallard and Pekin duck cells exposed to the same range of concentrations were up to 40 and 60 times greater than the corresponding responses of the chicken cells. The rates of 1-24 ACTH-induced B and DOC release from muscovy duck cells were similar to those from the Pekin duck cells; compared with the mallard duck cells, however, the muscovy duck cells were less sensitive and the maximum inducible rate of B release was significantly lower. The pattern of 1-24 ACTH-induced Aldo release from the muscovy duck cells was indistinguishable from that of the mallard duck cells, although the maximum inducible increase occurred at a lower concentration. Angiotensin II (AII) induced very small, but significant increased in B, Aldo, and DOC release from the chicken cells but in a nondose-related fashion. In contrast, mallard, Pekin, and muscovy duck cells all responded in a dose-dependent manner when incubated in medium containing AII. In each instance the maximum rate of Aldo synthesis induced by AII was about one-tenth of the corresponding rate induced by 1-24 ACTH. The maximum rates of B synthesis induced by AII, however, were extremely low compared with the rates induced by 1-24 ACTH. Thus, when maximally stimulated with AII the B:Aldo output ratio ranged from 1.0 to 1.9, whereas the corresponding ratio was between 9.0 and 17.0 when the cells were maximally stimulated with 1-24 ACTH.

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The morphological and functional changes associated with ageing were studied in adrenal steroidogenic cells derived from duck embryos. Cells grown for not more than three days had structural characteristics similar to their counterparts in vivo; they contained numerous lipid droplets and mitochondria, an abundant smooth endoplasmic reticulum, an even network of microtubules, and microfilaments that formed extensive and elaborate systems of parallel stress fibers. After the 3rd day of growth in culture, many of the cells started to decrease in size and become elongated; the older cells showed less well-defined actin filaments and contained elongated mitochondria, fewer lipid droplets, less smooth endoplasmic reticulum, and swollen cisternae of rough endoplasmic reticulum. The proliferative capacity of the cells was the same when they were cultured in either the presence or the absence of 1-24 ACTH. After the first day of growth in culture, the steroidogenic capacity of the cells declined and the addition of 1-24 ACTH to the growth medium did not prevent changes in their structure and function. The decline in steroidogenic capacity occurred both in terms of the amount of hormone released into the culture medium and in the ability of the cells to respond when incubated in buffer containing 1-24 ACTH. Since the basal unstimulated rates of corticosteroid production also declined as the cells aged, it is probable that the steroidogenic deficiency occurs at a site distal to the corticotropin receptor; this is also consistent with the ultrastructural observations that suggest a relationship between the morphological changes and the decline in steroidogenic capacity as the cells age.

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Cells derived from the adrenal glands of duck embryos immediately prior to hatching were grown in culture and used to study the morphological and cytoskeletal changes and steroidogenic responses induced by 1-24 ACTH. Changes in the cytoskeletal components were observed by rhodamine-phalloidin staining for actin and by staining the tubulin immunoreactive components with FITC. The cultures were comprised of a small population of chromaffin cells and a larger population of steroidogenic cells. The chromaffin cells were distinguished by their tyrosine hydroxylase immunoreactivity. The steroidogenic cells were characterized by the presence of sudanophilic lipid droplets, numerous mitochondria, abundant smooth endoplasmic reticulum, microtubules distributed as a fairly even network throughout the cytoplasm, and microfilaments that formed an extensive and elaborate system of stress fibers with many parallel arrays. The cells readily responded to stimulation with ACTH by releasing corticosterone, aldosterone and deoxycorticosterone. Stimulation with ACTH also induced changes in both the cell morphology and the cytoskeleton. Exposure of the cells to Krebs-Henseleit buffer containing 1-24 ACTH caused them to form numerous fine filopodia, to lose their stress fibers, and to form a thick ring of actin at the periphery of the cell. In addition, many cells became extremely arborized with many long branched dendritic processes. The morphological changes appeared to be related to a redistribution of the actin components, and may be explained only in part by the rounding up or retraction of the cytoplasm. The results strongly suggest an involvement of the actin components of the cytoskeleton in the steroidogenic response to corticotropic stimulation.

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The differentiating nephrotome in the 10-day-old mallard duck embryo is able to synthesize corticosterone, aldosterone and deoxycorticosterone even though an adrenal anlage cannot be identified histologically until the 12th day of incubation. At this time, sudanophilic cells containing much smooth endoplasmic reticulum and numerous mitochondria with tubular cristae are located adjacent to the developing mesonephros. Chromaffin cells appear in this region on about the 14th day of embryogenesis. A discrete glandular structure containing measurable quantities of corticosteroids can be identified on the 15th day, and during the next 2 days the tissue becomes encapsulated. Concomitantly, the ACTH-inducible rates of corticosteroid hormone synthesis increase several fold. The corticotropic responsiveness of the developing adrenal steroidogenic tissue increases progressively during the remainder of embryogenesis.

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1. Plasma concentrations of corticosterone (B), aldosterone (Aldo) and deoxycorticosterone (DOC) were measured in mallard ducklings immediately before and after exposure to acute immobilization stress. 2. Except for transient declines in B and DOC between the 4th and 14th days after hatching, the resting concentration of each hormone did not change significantly during post-natal development. 3. The stress-induced in Aldo was maximal at hatching while maximal increases in B and DOC did not occur until one day later. 4. Thereafter the magnitude of the stress-induced increases in the concentrations of all of the hormones decreased steadily and on the 21st and 28th days after hatching only B increased significantly in response to stress.

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1. Chronically colostomized ducks were injected with [4-14C]-aldosterone to study the metabolism of aldosterone and the pattern of metabolite excretion via the kidney. 2. Nearly half of the injected dose was excreted as radiometabolites during the first 24 hr; the largest amounts being excreted during the first 3 hr after injection. 3. Ion-exchange chromatography showed that monosulfate, disulfate, glucuronide, acidic, and neutral metabolites were excreted during each collection period, and that their relative proportions changed with time after injection of [4-14C]-aldosterone. 4. HPLC analysis of the neutral radiometabolites revealed 15 major peaks with retention times corresponding to both polar and reduced derivatives of aldosterone. 5. Only small quantities of unaltered labelled aldosterone were excreted. 6. Treatment of the birds with SKF 525-A caused a decrease in the total quantity of radiometabolite excreted and a change in the proportions of neutral and acidic metabolites in the cloacal fluid. 7. The decreases that occurred in the absolute amounts of some of the polar metabolites excreted by the birds treated with SKF-525A suggests that they may be hydroxylated and at least part of the aldosterone metabolizing system in the duck is cytochrome P450 dependent.

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1. Adjusting the Na+ and K+ intake of juvenile mallard ducks caused the plasma concentrations of corticosterone (B) and aldosterone (Aldo) to increase independently of one another, but none of these changes in electrolyte intake had a significant effect on the deoxycorticosterone (DOC) concentration. 2. With the exception of DOC in birds consuming the control diet, the plasma concentration of each hormone, regardless of diet, increased significantly following exposure to stress. 3. Stress-induced increases in Aldo concentration were greatest in birds given diets containing low concentrations of Na+. 4. Unlike the mammal and some other species of birds, Na+ may be the primary secretagogue responsible for the regulation of both corticosterone and aldosterone synthesis in the mallard duck.

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There are two regions of steroidogenic tissue in the bird adrenal gland: a subcapsular zone (SCZ) 40-60 cells thick consisting of cells with irregularly shaped nuclei, relatively little smooth endoplasmic reticulum, and mitochondria with shelflike cristae that surrounds an inner zone (IZ) of tissue comprised of smaller cells with rounded nuclei, a more abundant endoplasmic reticulum, and mitochondria with tubular cristae. The cristae in the mitochondria of IZ cells, but not the SCZ cells, seem to be quite labile and only assume the tubular configuration when subjected to corticotropic stimulation. The properties of the steroidogenic cells in the two zones are quantitatively distinct, the cells of the SCZ producing relatively more aldosterone and relatively less corticosterone than the cells of the IZ. The corticotropic responsiveness of the IZ cells is dependent on the synthesis of protein in the cytoplasm and a normal pattern of microfilament assembly. The dose responsiveness of both the IZ and the SCZ cells in vitro is a complex quadratic function of the corticotropin concentration to which they are exposed; the semilogarithmic linear response to corticotropin is restricted to a narrow midrange of concentrations, and at high concentrations cells from both zones of the gland respond submaximally. Throughout the dose-response range, however, the steroidogenic cells of the IZ are more sensitive and more responsive to corticotropic stimulation than are the cells of the SCZ.

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Tissues slices superfused with medium containing no ACTH released only traces of corticosterone. Addition of ACTH to the medium caused the rate of corticosterone release to increase to a maximum about 45 min after the addition of ACTH, after which time it either remained constant or started to wane slightly. The rate of release was affected by tissue thickness; the maximum rate of corticosterone release occurred when the tissue slices were 200 microns. Stimulated adrenocortical cells had large spherical nuclei, numerous mitochondria with tubular cristae, numerous lipid droplets, and a large amount of smooth endoplasmic reticulum. Many cells had an extensive network of microfilaments adjacent to the plasma membrane and some microtubules. Prolonged superfusion caused degenerative changes in some of the cells. Both cytochalasin B and cytochalasin D, dissolved in DMSO before addition to the superfusion medium, inhibited the corticotropic responsiveness in a dose-dependent manner. Control tissue samples superfused with medium containing DMSO, but no ACTH and no cytochalasin, released significantly more corticosterone than corresponding unstimulated samples. Few or no microfilaments were observed in adrenocortical cells after treatment with cytochalasin. Neither colchicine nor vinblastine had any discernible effect on the corticotropic responsiveness. After treatment with colchicine, adrenocortical cells had an ultrastructure characteristic of inner zone stimulated cells except that they were mainly devoid of microtubules.

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1. Doses of aldosterone (50, 100 and 200 micrograms per kg body wt) evoked similar changes in Na+ and K+ excretion by intact and colostomized ducks loaded with either distilled water or 0.5 isotonic saline (70 mM NaCl, 1.5 mM KCl); both antinatriuretic and antikaliuretic responses were observed. 2. The lowest dose of aldosterone had no effect on electrlyte excretion in intact and colostomized ducks loaded with a solution containing more K+ than Na+ (74 mM KCl, 36 mM NaCl) but the higher doses caused an antinatriuretic response in both groups; a retention of K+ occurred only in intact birds given this solution. 3. The lower dose of corticosterone (1.25 mg per kg body weight) caused both antidiuresis and antinatriuresis in intact birds, but in colostomized birds the decrease in Na+ excretion was not accompanied by an antidiuresis. 4. The higher dose of corticosterone (2.50 mg per kg body wt) caused a significant increase in K+ excretion in colostomized birds, whereas no kaliuresis was ever observed in intact birds. 5. Intact and colostomized birds loaded with 0.5 isotonic saline showed no responses to the lower dose of corticosterone, whereas the higher dose had an antikaliuretic effect in intact birds and an antinatriuretic effect in colostomized birds. 6. Corticosterone had no effect on cloacal water and electrolyte excretion by intact and colostomized birds given loads containing more K+ than Na+.

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1. The patterns of cloacal water and electrolyte excretion were qualitatively similar in intact and chronically colostomized ducks given oral loads of either distilled water or electrolyte solution. 2. The rates of Na+ and K+ excretion were each selectively increased in birds loaded with solutions containing a relatively higher concentration of each ion. 3. Compared to the intact birds, the colostomized birds showed lower rates of electrolyte excretion following administration of identical electrolyte loads and thus appeared to be retaining electrolyte. 4. At the same time, a significant antidiuresis of similar magnitude occurred in each group of saline-loaded birds. 5. The possible contributions of renal and post-renal mechanisms to the overall conservation of water in intact and colostomized ducks are discussed.

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Unstressed mallard ducks (anas platyrhychos), given uncontaminated food and maintained on a short photoperiod, show two daily maxima in plasma corticosterone concentration ([B]) on occurring early in the light phase and a second just before the onset of darkness. After one week of exposure to food containing 3% (v/w) South Louisiana crude oil, plasma [B] were significantly lowered throughout the day. Similar abrupt declines in plasma [B] also occurred during the first 10 days of exposure to food containing 1% and 0.5% crude oil. Although the plasma [B] in birds consuming food contaminated with 0.5% crude oil increased between 10 and 50 days of exposure, the concentration after 50 days was still lower than normal. During the same interval, normal plasma [B] were restored in birds consuming food containing 1% and 3% crude oil. Significant increases occurred in the naphthalene-metabolizing properties of hepatic microsomes prepared from birds acutely exposed to all levels of petroleum-contaminated food and elevated levels were sustained throughout the first 50 days of exposure. Birds given food containing 3% crude oil for more than 50 days, however, showed steady declines in hepatic naphthalene-metabolizing activity. After 500 days, the activity was similar to that found in contemporaneous controls. During the same interval the plasma [B] increased until the levels were higher than normal after 500 days of exposure; at this time, an inverse relationship, similar to that seen during the first week of exposure to contaminated food, was once more established between plasma [B] and the concomitant hepatic naphthalene-metabolizing activity.

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